Thrombin is an important serine protease in hemostasis and thrombosis, and is present in blood plasma in the form of a precursor, pro-thrombin. One of the key actions of thrombin is cellular modulation via receptor activation. A functional human thrombin receptor (PAR-1), which was cloned in 1991 (Vu, T. K., et al., Cell 64(6):1057-68 (1991)), was found to be a member of the G-protein coupled receptor (GPCR) superfamily. Receptor activation of PAR-1 putatively occurs by N-terminal recognition and proteolytic cleavage at the Arg-41/Ser-42 peptide bond to reveal a truncated N-terminus. This new N-terminus, acting as a tethered ligand to recognize a site on the receptor, can trigger activation and signal transduction leading to platelet aggregation. Since 1991, three other protease-activated receptors with extensive homology to this thrombin receptor, namely “PAR-2” (Nystedt, S., et al., Proc. Natl. Acad. Sci. USA 91:9208-12 (1994)), “PAR-3” (Ishihara, H., et al., Nature 386(6624):502-06 (1997)), and “PAR-4” (Xu, W. F., et al., Proc. Natl. Acad. Sci. USA 95(12):6642-46 (1998)), have been cloned. Thrombin receptor (PAR-1) specific antibody-induced blockade of the platelet thrombin receptor has shown efficacy against arterial thrombosis in vivo (Cook, J. J., et al., Circulation 91(12):2961-71 (1995)). Hence, antagonists of the thrombin receptor (PAR-1) are useful to block these protease-activated receptors and, as such, may be used to treat platelet-mediated thrombotic disorders, such as myocardial infarction, stroke, restenosis, angina, atherosclerosis, and ischemic conditions.
Although particular protease-activated receptors (e.g., PAR-1, PAR-2, PAR-3 and PAR-4) have been identified, thrombin also possesses growth-promoting activity for a wide variety of cells from various tissues, which results from activation of a specific cell surface receptor known as the non-proteolytically activated thrombin receptor (NPAR) (Horvat, R., et. al., J. Cell Sci. 108, 1155-64 (1995)). For example, thrombin has been shown to promote angiogenesis, the development of new blood vessels, and to stimulate endothelial cell proliferation (see, e.g., U.S. Pat. Nos. 5,352,664 and 5,500,412; the contents of both of which are incorporated herein by reference in their entirety). To date, this NPAR has not been cloned or characterized at the molecular level.
The discovery that protein sequences encoded by complementary strands of nucleic acids can bind to one another is known as the molecular recognition theory (MRT) (Blalock, Nature Medicine 1:876-878 (1995)). This theory, which is based on the development of complementary peptides (CPs) specified by ligand antisense RNA, has proven useful in designing interactive peptides, isolating receptors, and producing anti-receptor and anti-idiotypic antibodies (see, e.g., Blalock, et al., Trends Biotechnol. 8:140-144 (1990); and Clarke, et al., In: Antisense Nucleic Acids and Proteins: Fundamentals and Applications vandrol Krol, A. V., and Mol, J. N. M. (eds): Dekker, New York: 169-186 (1991)). Antibodies to particular CPs have been used to identify specific receptors that have been difficult to purify.
Given the importance of thrombin in such diverse processes as mitogenesis, cell growth, wound healing, tissue adhesion, angiogenesis and tumor metastasis, it would be useful to have new molecules that can stimulate or inhibit one or more thrombin functions. It would also be useful to identify NPAR, which is expressed on a wide variety of cells, and is involved in thrombin-induced growth-promoting activity.